Team:Brown/Notebook/July7

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Wednesday, July 7 2010

Results of overnight plating:

  • Empty pNoTat showed <5 colonies (suspect)
  • Religated pGEM, pNoTat showed no growth.
  • Control RFP showed colonies

Harvested 4 colonies from empty pNoTat plate.

Added to 5 mL of LB in culture tube, added 10 µl of Amp (100 mg/mL) to achieve total 200 µl/mL

pWill1 Glycerol Stocks

Added 750 µl culture, 250 µl glycerol per tube.

2 overnight tubes (1 and 2) stocked into 2 cyro tubes each.

LB Agar plates

  • Mixed 12.5 g LB lennox broth with 7.5 g Agar and 500 mL dH2O
  • Mixed 6.25 g LB lennox broth with 3.25 g Agar in 250 mL dH2O
  • Cooked both until clear (not sure about concentrations)

Redo Ligation of WillRS and pGEM

  • 7.5 µl 2X rapid ligation buffer
  • 1.0 µl T4 DNA ligase
  • 0.5 µl pGEM T Easy Vector
  • 1.5 µl Will1 RS insert
  • 4.5 µl dH2O

Made competent CaCl2 cells (Quick protocol)

Transferred Xl1 cells with ligation

Plated on amp+ plates, incubate at 37°C starting 7:00 PM